PROJECT SUMMARY Most internal organs consist of a monolayer of polarized epithelial cells surrounding a central lumen that establish a barrier that segregate the internal medium from the outside environment. One of the key determinants of establishment of apicobasal polarity in epithelial cells is the Scribble complex. The Scribble complex is a multi- protein scaffolding platform which functions by recruiting other binding partners, to build spatially distinct signaling complexes. A major barrier to understand the formation of the epithelium is our poor understanding of the mechanisms that control the function of the Scribble complex. For over two decades, the Scribble complex was known to function as a module from genetic studies, but it was unclear how the members of the complex interacted with each other, and their link to the regulators of adhesion and polarity. To close this gap, my lab is focused in defining the precise composition, mechanism of formation and regulation of the Scribble complex. We have recently shown that SGEF, a RhoG-specific GEF, is a new component of the Scribble complex that acts as a scaffold to form a ternary complex by interacting directly with Scribble and Dlg1, and plays a role during junction formation, apical contractility, E-cadherin stability and lumen formation. Based on these findings, the objective of this proposal is to define the molecular mechanisms that regulate the function of the Scribble complex in the regulation of cell-cell junctions? assembly and maintenance, and lumen formation. We will test the central hypothesis that the incorporation of SGEF into the Scribble complex stimulates its catalytic activity, which in turn activates RhoG to regulate the stability of cell-cell junctions, as well as lumen opening and number. We propose the following specific aims to test this hypothesis. Aim 1. Define the spatiotemporal regulation of SGEF and RhoG activity by the Scribble complex. Here we will test our working hypothesis that binding to Scribble and Dlg1 targets and/or activates SGEF to cell-cell junctions which promote the localized activation of RhoG; Aim 2. Determine the mechanism by which the Scribble/SGEF/Dlg1 complex regulates E-cadherin stability. Here we will test our working hypothesis that the activation of RhoG downstream SGEF stabilizes E-cadherin at the membrane by regulating its recycling and/or degradation; Aim 3. Determine how the Scribble/SGEF/Dlg1 complex regulates lumen opening and number. Here, we will test our working hypothesis that, the formation of the Scribble/SGEF/Dlg1 is required the formation of cysts with a single, central open lumen in 3D cysts. This approach is innovative, as it provides mechanistic insight on the function of the Scribble complex. This contribution will be significant because it will shed light on the fundamental mechanisms controlling cell-cell adhesion and its role in the establishment of in epithelial cells and tissues. Understanding how cell adhesion and polarity are established and maintained in normal cells is important for situations in which loss of adhesion and polarity represent a problem, such as epithelial cancers, which comprise 85% of all human cancers.